All lanes : Anti-HA tag antibody ( ab13834 ) at 1/500 dilution Lane 1 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate - expressing HA tag protein ( ab20896 ) Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate - expressing HA tag protein ( ab20896 ) with HA tag peptide (ab13835) at 1 µg/ml Lysates/proteins at 10 µg per lane. Secondary Alexa Fluor 680 Goat-anti-Rabbit IgG at 1/5000 dilution Performed under reducing conditions. Observed band size : 55 kDa ( why is the actual band size different from the predicted? )